This research proposal looks at the importance of viral diversity on transmission and evolution. Viruses exist not as clonal populations but as 'swarms' of variants with mutations along the genome. Variation has not been characterised in arboviruses (arthropod-borne viruses), which require infection of a vertebrate host and a vector (such as a mosquito or tick). During infection of the mosquito there are several anatomical barriers that reduce the number of viral particles, thus a bottleneck is imposed on the virus. It is hypothesised that, upon infection of the mosquito vector, the amount of variation decreases, leading to a decreased rate of evolution in the arboviruses as compared to viruses with a single host. The rate of evolution is important to understand viral ecology and emergence. Venezuelan equine encephalitis virus (VEEV) and two natural mosquito vectors will be used to model viral variation present in wild-type transmission, and the impact that mosquito infection has on viral diversity. VEEV is of interest because it is both an important, naturally emerging virus and a potential bioweapon with no licensed vaccine or therapy. The virus circulates in Mexico and has caused outbreaks in equids and humans in the U.S. Viral diversity will be investigated using 3 specific aims. First, the bottleneck profile of the epidemic vector Psorophora confinnis will be determined compared to the bottleneck profile already determined for the enzootic vector Culex taeniopus. Secondly, the profile of intra-host variation of VEEV subtypes IE and IC will be determined upon infection of the mosquito vectors C. taeniopus and Aedes taeniorhynchus and an appropriate rodent host, individually and as part of a transmission cycle. The effect of the anatomical mosquito bottlenecks on viral diversity will be investigated using an established protocol to initiate a laboratory transmission cycle that approximates to that occurring naturally. The levels of diversity will be determined using Next Generation Sequencing technology to determine the minority populations present throughout the transmission cycle. This decrease in diversity may lead to a reduction in fitness due to stochastic effects. The level of drift caused by this decrease in diversity will be tested via an experimental transmission cycle. Thirdly, the change in the ability of the virus to complete a transmission cycle when the polymerase fidelity is increased by genetic manipulation will be examined. It is hypothesized that viral variation aids in viral survival therefore viruses mutated to exhibit high-fidelity in their replication will be used in experimental infections. It is expected that viral survival will decrease. The results of this project will enhance our understanding of arboviral population genetic dynamics occurring in the mosquito vector as well as determine the degree to which natural selection and genetic drift influence the persistence and emergence of new arboviruses. The results will translate to other emerging arboviruses such as dengue, West Nile and chikungunya, as well as provide the data to generate effective models of arbovirus emergence.